Mutations in SOX9, the Gene Responsible for Campomelic Dysplasia and Autosomal Sex Reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.     

Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions.

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-coding genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F. rubripes. In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available, mouse sequences confirm conservation of the elements. From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.     

Autosomal recessive disorder otospondylomegaepiphyseal dysplasia is associated with loss-of-function mutations in the COL11A2 gene

Otospondylomegaepiphyseal dysplasia (OSMED) is an autosomal recessive skeletal dysplasia accompanied by severe hearing loss. The phenotype overlaps that of the autosomal dominant disorders-Stickler and Marshall syndromes-but can be distinguished by disproportionately short limbs, severe hearing loss, and lack of ocular involvement. In one family with OSMED, a homozygous Gly-->Arg substitution has been described in COL11A2, which codes for the alpha2 chain of type XI collagen. We report seven further families with OSMED. All affected individuals had a remarkably similar phenotype: profound sensorineural hearing loss, skeletal dysplasia with limb shortening and large epiphyses, cleft palate, an extremely flat face, hypoplasia of the mandible, a short nose with anteverted nares, and a flat nasal bridge. We screened affected individuals for mutations in COL11A2 and found different mutations in each family. Individuals from four families, including three with consanguineous parents, were homozygous for mutations. Individuals from three other families, in whom parents were nonconsanguineous, were compound heterozygous. Of the 10 identified mutations, 9 are predicted to cause premature termination of translation, and 1 is predicted to cause an in-frame deletion. We conclude that the OSMED phenotype is highly homogenous and results from homozygosity or compound heterozygosity for COL11A2 mutations, most of which are predicted to cause complete absence of alpha2(XI) chains.     

Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca²⁺ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca²⁺ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.     

Deletions in the COL10A1 gene are not associated with skeletal changes in dogs

Type 10 collagen alpha 1 (COL10A1) is a short-chain collagen of cartilage synthesized by chondrocytes during the growth of long bones. COL10A1 mutations, which frequently result in COL10A1 haploinsufficiency, have been identified in patients with Schmid metaphyseal chondrodysplasia (SMCD), a cartilage disorder characterized by short-limbed short stature and bowed legs. Similarities between SMCD and short stature in various dog breeds suggested COL10A1 as a candidate for canine skeletal dysplasia. We report the sequencing of the exons and promoter region of the COL10A1 gene in dog breeds fixed for a specific type of skeletal dysplasia known as chondrodysplasia, breeds that segregate the skeletal dysplasia phenotype, and control dogs of normal stature. Thirteen single nucleotide polymorphisms (SNPs), one insertion, and two deletions, one of which introduces a premature stop codon and likely results in nonsense-mediated decay and the degradation of the mutant allele product, were identified in the coding region. There appear to be no causal relationships between the polymorphisms identified in this study and short stature in dogs. Although COL10A1 haploinsufficiency is an important cause of SMCD in humans, it does not seem to be responsible for the skeletal dysplasia phenotype in these dog breeds. In addition, homozygosity for the nonsense allele does not result in the observed canine skeletal dysplasia phenotype. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Mutations in three subdomains of the carboxy-terminal region of collagen type X account for most of the Schmid metaphyseal dysplasias

We have used the polymerase chain reaction and single strand conformation polymorphism (SSCP) methods to analyse the COL10A1 gene, which encodes collagen type X, in DNA samples from patients with metaphyseal dysplasia type Schmid (SMCD) and other related forms of metaphyseal dysplasia. Five cases of SMCD were sporadic and three others were familial. Abnormal SSCP profiles were observed in six instances. In two families, the altered pattern segregated with the phenotype. The heterozygous mutations corresponded to a glycine substitution by glutamic acid at position 595 and to an asparagine substitution by lysine at position 617. In one sporadic case, the sequence studies demonstrated that the individual was heterozygous for a single base deletion (del T 1908) that produced a premature stop codon. Three additional mutations were single base substitutions that affected highly conserved residues at positions 597, 644 and 648. In two additional individuals with SMCD, in two patients with unclassifiable forms of metaphyseal dysplasia, and in one family with epiphyso-metaphyseal dysplasia, SSCP analysis detected neutral polymorphisms in the entire coding sequence of the gene but no mutations. Our results demonstrate that mutations in the carboxy-terminal region of collagen X are specific for the SMCD phenotype. Mutations appear to be clustered into three small subdomains: one of them is rich an aromatic residues, the second includes the putative N-linked oligosaccharide attachment site and the third contains mostly hydrophilic residues. The absence of clinical variability between patients carrying heterozygous single base substitutions or small deletions suggests that, in both instances, the mutant collagen chains either fail to be incorporated into stable trimers or disturb type X collagen assembly.     

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.     

Normal lung development and function after Sox9 inactivation in the respiratory epithelium

Heterozygous mutations in the human SOX9 gene cause campomelic dysplasia (CD), a skeletal malformation syndrome with various other organ defects. Severely affected CD patients usually die in the neonatal period due to respiratory distress. We analyzed the dynamic expression pattern of Sox9 in the developing mouse lung throughout morphogenesis. To determine a role of Sox9 in lung development and function, Sox9 was specifically inactivated in respiratory epithelial cells of the mouse lung using a doxycycline-inducible Cre/loxP system. Immunohistochemical and RNA analysis demonstrated extensive inactivation of Sox9 in the embryonic stage of lung development as early as embryonic day (E) 12.5. Lung morphogenesis and lung function after birth were not altered. Compensatory upregulation of Sox2, Sox4, Sox8, Sox10, Sox11, and Sox17 was not detected. Although Sox9 is expressed at high levels throughout lung morphogenesis, inactivation of Sox9 from the respiratory epithelial cells does not alter lung structure, postnatal survival, or repair following oxygen injury.     

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.     

SOX10 mutations in chronic intestinal pseudo-obstruction suggest a complex physiopathological mechanism

The type IV Waardenburg syndrome (WS4), also referred to as Shah-Waardenburg syndrome or Waardenburg-Hirschsprung disease, is characterised by the association of Waardenburg features (WS, depigmentation and deafness) and the absence of enteric ganglia in the distal part of the intestine (Hirschsprung disease). Mutations in the EDN3, EDNRB, and SOX10 genes have been reported in this syndrome. Recently, a new SOX10 mutation was observed in a girl with a neural crest disorder without evidence of depigmentation, but with severe constipation due to a chronic intestinal pseudo-obstruction and persistence of enteric ganglia. To refine the nosology of WS, we studied patients with typical WS4 (including Hirschsprung disease) or with WS and intestinal pseudo-obstruction. We found three SOX10 mutations, one EDNRB and one EDN3 mutations in patients presenting with the classical form of WS4, and two SOX10 mutations in patients displaying chronic intestinal pseudo-obstruction and WS features. These results show that chronic intestinal pseudo-obstruction may be a manifestation associated with WS, and indicate that aganglionosis is not the only mechanism underlying the intestinal dysfunction of patients with SOX10 mutations.     

Neurological phenotype in Waardenburg syndrome type 4 correlates with novel SOX10 truncating mutations and expression in developing brain

Waardenburg syndrome type 4 (WS4), also called Shah-Waardenburg syndrome, is a rare neurocristopathy that results from the absence of melanocytes and intrinsic ganglion cells of the terminal hindgut. WS4 is inherited as an autosomal recessive trait attributable to EDN3 or EDNRB mutations. It is inherited as an autosomal dominant condition when SOX10 mutations are involved. We report on three unrelated WS4 patients with growth retardation and an as-yet-unreported neurological phenotype with impairment of both the central and autonomous nervous systems and occasionally neonatal hypotonia and arthrogryposis. Each of the three patients was heterozygous for a SOX10 truncating mutation (Y313X in two patients and S251X [corrected] in one patient). The extended spectrum of the WS4 phenotype is relevant to the brain expression of SOX10 during human embryonic and fetal development. Indeed, the expression of SOX10 in human embryo was not restricted to neural-crest-derived cells but also involved fetal brain cells, most likely of glial origin. These data emphasize the important role of SOX10 in early development of both neural-crest-derived tissues, namely melanocytes, autonomic and enteric nervous systems, and glial cells of the central nervous system.     

Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.